Human parainfluenza viruses (HPIVs) are important pathogens in human populations, causing severe lower respiratory tract infections in infants and young children. HPIV1 and HPIV2 are the principal etiologic agents of laryngotracheobronchitis (croup), and can also cause pneumonia and bronchiolitis (Chanock et al., Parainfluenza Viruses., p. 1341-1379, In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E. Straus (eds.) Fields Virology, 4th ed., Vol. 1, Lippincott Williams & Wilkins, Philadelphia, 2001). HPIV3 ranks second after respiratory syncytial virus (RSV) as a leading cause of hospitalization for viral lower respiratory tract disease in infants and young children (Collins et al., 3rd ed. In “Fields Virology,” B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1205-1243. Lippincott-Raven Publishers, Philadelphia, 1996; Crowe et al., Vaccine 13:415-421, 1995; Marx et al., J. Infect. Dis. 176:1423-1427, 1997).
HPIVs are also important causes of respiratory tract disease in adults. Collectively, HPIV1, HPIV2, and HPIV3 have been identified through a 20 year study as responsible etiologic agents for approximately 18% of hospitalizations for pediatric respiratory tract disease (Murphy et al., Virus Res. 11:1-15, 1988). HPIVs have also been implicated in a significant proportion of cases of virally-induced middle ear effusions in children with otitis media (Heikkinen et al., N. Engl. J. Med. 340:260-4, 1999).
Despite considerable efforts to develop effective vaccines against HPIVs, no vaccines have yet been approved for any HPIV serotype, nor for ameliorating HPIV related illnesses. The most promising prospects to date are live attenuated vaccine viruses since these have been shown to be efficacious in non-human primates even in the presence of passively transferred antibodies, an experimental situation that simulates that present in the very young infant who possesses maternally acquired antibodies (Crowe et al., Vaccine 13:847-855, 1995; Durbin et al., J. Infect. Dis. 179:1345-1351, 1999). Two live attenuated HPIV3 vaccine candidates, a temperature-sensitive (ts) derivative of the wild type HPIV3 JS strain (designated HPIV3cp45) and a bovine PIV3 (BPIV3) strain, are undergoing clinical evaluation (Karron et al., Pediatr. Infect. Dis. J. 15:650-654, 1996; Karron et al., J. Infect. Dis. 171:1107-1114, 1995a; Karron et al., J. Infect. Dis. 172, 1445-1450, 1995b). The live attenuated PIV3cp45 vaccine candidate was derived from the JS strain of HPIV3 via serial passage in cell culture at low temperature and has been found to be protective against HPIV3 challenge in experimental animals and to be satisfactorily attenuated, genetically stable, and immunogenic in seronegative human infants and children (Belshe et al, J. Med. Virol. 10:235-242, 1982; Belshe et al., Infect. Immun. 37:160-5, 1982; Clements et al., J. Clin. Microbiol. 29:1175-82, 1991; Crookshanks et al., J. Med. Virol. 13:243-9, 1984; Hall et al., Virus Res. 22:173-184, 1992; Karron et al., J. Infect. Dis. 172:1445-1450, 1995b). Because these HPIV3 candidate vaccine viruses are biologically derived, there are no proven methods for adjusting the level of attenuation should this be found necessary from ongoing clinical trials.
To facilitate development of HPIV vaccines, recombinant DNA technology has recently made it possible to recover infectious negative-stranded RNA viruses from cDNA (for a review, see Conzelmann, J. Gen. Virol. 77:381-89, 1996). In this context, recombinant rescue of infectious virus has been reported for respiratory syncytial virus (RSV), rabies virus (RaV), canine distemper virus, mumps virus, infectious hematopoietic necrosis virus, simian virus 5 (SV5), rinderpest virus, Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), measles virus (MeV), and Sendai virus (murine parainfluenza virus type 1 (MPIV1)) from cDNA-encoded genomic or antigenomic RNA in the presence of essential viral proteins (see, e.g., Garcin et al., EMBO J. 14:6087-6094, 1995; Lawson et al., Proc. Natl. Acad. Sci. U.S.A. 92:4477-81, 1995; Radecke et al., EMBO J. 14:5773-5784, 1995; Schnell et al., EMBO J. 13:4195-203, 1994; Whelan et al., Proc. Natl. Acad. Sci. U.S.A. 92:8388-92, 1995; Hoffman et al., J. Virol. 71:4272-4277, 1997; Kato et al., Genes to Cells 1:569-579, 1996, Roberts et al., Virology 247:1-6, 1998; Baron et al., J. Virol. 71:1265-1271, 1997; International Publication No. WO 97/06270; Collins et al., Proc. Natl. Acad. Sci. U.S.A. 92:11563-11567, 1995; Clarke et al., J. Virol. 74:4831-4838, 2000; Biacchesi et al., J. Virol. 74:11247-11253, 2000; Gassen et al., J. Virol. 74:10737-10744, 2000; U.S. patent application Ser. No. 08/892,403, filed Jul. 15, 1997 (corresponding to published International Application No. WO 98/02530 and priority U.S. Provisional Application Nos. 60/047,634, filed May 23, 1997, 60/046,141, filed May 9, 1997, and 60/021,773, filed Jul. 15, 1996); U.S. patent application Ser. No. 09/291,894, filed on Apr. 13, 1999; International Application No. PCT/US00/09695, filed Apr. 12, 2000 (which claims priority to U.S. Provisional Patent Application Ser. No. 60/129,006, filed Apr. 13, 1999); International Application No. PCT/US00/17755, filed Jun. 23, 2000 (which claims priority to U.S. Provisional Patent Application Ser. No. 60/143,132, filed by Buchholz et al. on Jul. 9, 1999); Juhasz et al., J. Virol. 71:5814-5819, 1997; He et al. Virology 237:249-260, 1997; Peters et al. J. Virol. 73:5001-5009, 1999; Baron et al. J. Virol. 71:1265-1271, 1997; Whitehead et al., Virology 247:232-9, 1998a; Whitehead et al., J. Virol. 72:4467-4471, 1998b; Jin et al. Virology 251:206-214, 1998; Buchholz et al. J. Virol. 73:251-259, 1999; and Whitehead et al., J. Virol. 73:3438-3442, 1999, each incorporated herein by reference in its entirety for all purposes).
Additional publications in the field of the invention report recovery of recombinant parainfluenza viruses (PIVs), specifically HPIV1, HPIV2, HPIV3, and BPIV3 (see, e.g., Durbin et al., Virology 235:323-332, 1997; Schmidt et al., J. Virol. 74:8922-8929, 2000; Kawano et al., Virology 284:99-112, 2001; U.S. patent application Ser. No. 09/083,793, filed May 22, 1998 (corresponding to U.S. Provisional Application No. 60/059,385, filed Sep. 19, 1997); U.S. Provisional Application No. 60/331,961, filed Nov. 21, 2001; and U.S. Provisional Application No. 60/047,575, filed May 23, 1997 (corresponding to International Publication No. WO 98/53078), each incorporated herein by reference). Some of these reports further address genetic manipulation of viral cDNA clones to determine the genetic basis of phenotypic changes in biological mutants, for example, which mutations in a biological mutant HPIV3 JS cp45 virus specify its ts, ca and att phenotypes, and which gene(s) or genome segment(s) of BPIV specify its attenuation phenotype. Additionally, certain of these reports and related publications discuss construction of novel PIV vaccine candidates having a wide range of different mutations, as well as methods for evaluating the level of attenuation, immunogenicity and phenotypic stability exhibited by such recombinant vaccine candidates (see also, U.S. application Ser. No. 09/586,479, filed Jun. 1, 2000 (corresponding to U.S. Provisional Patent Application Ser. No. 60/143,134, filed on Jul. 9, 1999); and U.S. patent application Ser. No. 09/350,821, filed by Durbin et al. on Jul. 9, 1999, each incorporated herein by reference).
Thus, infectious wild type recombinant PIVs, (r)PIVs, as well as a number of ts and otherwise modified derivatives, have now been recovered from cDNA. Reverse genetics systems have been used to generate infectious virus bearing defined mutations that specify attenuation and other desirable phenotypes, and to study the genetic basis of attenuation and other phenotypic changes in existing vaccine candidate viruses. For example, in HPIV3, the three amino acid substitutions found in the L gene of cp45, singularly or in combination, have been found to specify the ts and attenuation phenotypes. Additional ts and other attenuating mutations can be introduced in other regions of the HPIV3 genome.
In addition, a chimeric PIV1 vaccine candidate has been generated using the PIV3 cDNA rescue system by replacing the PIV3 HN and F open reading frames (ORFs) with those of PIV1 in a PIV3 full-length cDNA, that optionally contains selected attenuating mutations. Exemplary recombinant chimeric viruses derived from these cDNA-based methods include a HPIV3-1 recombinant bearing all three identified mutations in the L gene, rPIV3-1.cp45L (Skiadopoulos et al., J. Virol. 72:1762-8, 1998; Tao et al., J. Virol. 72:2955-2961, 1998; Tao et al., Vaccine 17:1100-1108, 1999, incorporated herein by reference). rHPIV3-1.cp45L was attenuated in hamsters and induced a high level of resistance to challenge with HPIV1. Yet another recombinant chimeric virus, designated rHPIV3-1cp45, has been produced that contains 12 of the 15 cp45 mutations, i.e., excluding the mutations that occur in HN and F. This recombinant vaccine candidate is highly attenuated in the upper and lower respiratory tract of hamsters and non-human primates and induces a high level of protection against HPIV1 infection (Skiadopoulos et al., Vaccine 18:503-510, 1999; Skiadopoulos et al., Virology 297:136-152, 2002, each incorporated herein by reference). However, for use against HPIV1, the infection and attendant immunogenicity of chimeric HPIV3-1 vaccine candidates against HPIV1 challenge is dampened in hosts that exhibit immune recognition of HPIV3.
Recently, a number of studies have focused on the possible use of viral vectors to express foreign antigens toward the goal of developing vaccines against a pathogen for which other vaccine alternatives have not yet proven successful. In this context, a number of reports suggest that foreign genes may be successfully inserted into a recombinant negative strand RNA virus genome or antigenome with varying effects (Bukreyev et al., J. Virol. 70:6634-41, 1996; Bukreyev et al., Proc. Natl. Acad. Sci. U.S.A. 96:2367-72, 1999; Finke et al. J. Virol. 71:7281-8, 1997; Hasan et al., J. Gen. Virol. 78:2813-20, 1997; He et al., Virology 237:249-60, 1997; Jin et al., Virology 251:206-14, 1998; Johnson et al., J. Virol. 71:5060-8, 1997; Kahn et al., Virology 254:81-91, 1999; Kretzschmar et al., J. Virol. 71:5982-9, 1997; Mebatsion et al., Proc. Natl. Acad. Sci. U.S.A. 93:7310-4, 1996; Moriya et al., FEBS Lett. 425:105-11, 1998; Roberts et al., J. Virol. 73:3723-32, 1999; Roberts et al., J. Virol. 72:4704-11, 1998; Roberts et al., Virology 247:1-6, 1998; Sakai et al., FEBS Lett. 456:221-226, 1999; Schnell et al., Proc. Natl. Acad. Sci. U.S.A. 93:11359-65, 1996a; Schnell et al., J. Virol. 70:2318-23, 1996b; Schnell et al., Cell 90:849-57, 1997; Singh et al., J. Gen. Virol. 80:101-6, 1999; Singh et al., J. Virol. 73:4823-8, 1999; Spielhofer et al., J. Virol. 72:2150-9, 1998; Yu et al., Genes to Cells 2:457-66 et al., 1999; Duprex et al., J. Virol. 74:7972-7979, 2000; Subash et al., J. Virol. 74:9039-9047, 2000; Krishnamurthy et al., Virology 278:168-182, 2000; Rose et al., J. Virol. 74:10903-10910, 2000; Tao et al., J. Virol. 74:6448-6458, 2000; McGettigan et al., J. Virol. 75:8724-8732, 2001; McGettigan et al., J. Virol. 75:4430-4434, 2001; Kahn et al., J. Virol. 75:11079-11087, 2001; Stope et al., J. Virol. 75:9367-9377, 2001; Huang et al., J. Gen. Virol. 82:1729-1736, 2001; Skiadopoulos et al., J. Virol. 75:10498-10504, 2001; Bukreyev et al., J. Virol. 75:12128-12140, 2001; U.S. patent application Ser. No. 09/614,285, filed Jul. 12, 2000 (corresponding to U.S. Provisional Patent Application Ser. No. 60/143,425, filed on Jul. 13, 1999), each incorporated herein by reference). When inserted into the viral genome under the control of viral transcription gene-start and gene-end signals, the foreign gene may be transcribed as a separate mRNA and yield significant protein expression. Surprisingly, in most cases the foreign sequence has been reported to be relatively stable and capable of expressing functional protein during numerous passages in vitro.
In order to successfully develop vectors for vaccine use, however, it is insufficient to simply demonstrate a high, stable level of protein expression. For example, this has been possible since the early-to-mid 1980s with recombinant vaccinia viruses and adenoviruses, and yet these vectors have proven to be disappointing tools for developing vaccines for human use. Similarly, most nonsegmented negative strand viruses that have been developed as vectors have not yet been shown to be amenable for human vaccine use. Examples in this context include vesicular stomatitis virus, an ungulate pathogen with no history of administration to humans except for a few laboratory accidents; Sendai virus, a mouse pathogen with no history of administration to humans; simian virus 5, a canine pathogen with no history of intentional administration to humans; and an attenuated strain of measles virus which must be administered systemically and would be neutralized by measles-specific antibodies present in nearly all humans due to maternal antibodies and widespread use of a licensed measles vaccine. Furthermore, some of these prior vector candidates have adverse effects, such as immunosuppression, which are directly inconsistent with their use as vectors. Thus, one must identify vectors whose growth characteristics, tropisms, and other biological properties make them appropriate as vectors for human use. It is further necessary to develop a viable immunization strategy, including efficacious timing and route of administration.
Proposed mononegaviruses for use as vaccine vectors include measles, mumps, VSV, and rabies viruses. However, measles virus has limitations relating to its potential use as a vaccine vector. For example, measles virus has been considered for use a vector for the protective antigen of hepatitis B virus (Singh et al., J. Virol. 73:4823-8, 1999). However, this combined measles virus-hepatitis B virus vaccine candidate could only be administered after nine months of age, on a schedule comparable to the indicated schedule for the licensed measles virus vaccine, whereas the current hepatitis B virus vaccine is recommended for use in early infancy. This is because the currently licensed measles vaccine is administered parenterally and is sensitive to neutralization and immunosuppression by maternal antibodies, and therefore is not effective if administered before 9-15 months of age. Thus, measles virus is a poor vector for antigens of pathogenic agents that cause disease in early infancy, such as RSV and the HPIVs.
The attenuated measles virus vaccine has been associated with altered immune responses and excess mortality when administered at increased dosages, which may be due at least in part to virus-induced immunosuppression that is a common feature of natural measles virus infection. This indicates that even an attenuated measles virus may not be suitable for vaccine vector use. Furthermore, the use of measles virus as a vector would be inconsistent with the global effort to eradicate this pathogen. Indeed, for these reasons it would be desirable to end the use of live measles virus and replace the present measles virus vaccine with a suitable non-measles vector that expresses measles virus protective antigens.
Rabies virus, a rare cause of infection of humans, has been considered for use as a vector (Mebatsion et al., Proc. Natl. Acad. Sci. USA 93:7310-4, 1996), but it is unlikely that a virus that is so highly fatal as rabies for humans could be developed for use as a live attenuated virus vector. Moreover, immunity to the rabies virus, which is not a ubiquitous human pathogen, is not needed for the general population. In addition, in some circumstances it may be desirable for the vector to be capable of eliciting a multispecific immune response against both the vector virus and the pathogen for which the vector is used as a carrier of antigenic determinants. While measles virus is less pathogenic than the rabies virus, infection by either of this vector candidate can yield undesirable results. Measles virus establishes a viremia with widespread infection and associated rash and the above-mentioned immunosuppression. Mild encephalitis during measles infection is not uncommon. Measles virus is also associated with a rare progressive fatal neurological disease called subacute sclerosing encephalitis.
In contrast to such vector candidates as rabies and measles, PIV infection and disease is typically more limited, in part by confinement of infection to the respiratory tract. Viremia and spread to secondary sites can occur in severely immunocompromised subjects, but this is not a typical effect of PIV infection. Acute respiratory tract disease is the only disease associated with PIVs. Thus, the use of PIVs as vectors will, on the basis of their biological characteristics, avoid complications such as interaction of virus with peripheral lymphocytes, leading to immunosuppression, or infection of secondary organs such as the testes or central nervous system, leading to other complications. These characteristics also render PIV a better vector candidate for successful immunization, which can be achieved more easily and effectively via alternate routes, such as direct administration to the respiratory tract, compared to immunization with vectors that require parental administration.
Among a host of human pathogens for which a vector-based vaccine approach may be desirable is the measles virus. A live attenuated vaccine has been available for more than three decades and has been largely successful in eradicating measles disease in the United States. However, the World Health Organization estimates that more than 45 million cases of measles still occur annually, particularly in developing countries, and the virus contributes to approximately one million deaths per year. One reason for the persistence of this disease is the inefficacy of current vaccine formulations to overcome maternal antibodies that inactivate the current vaccine.
Measles virus is a member of the Morbillivirus genus of the Paramyxoviridae family (Griffin et al., In “Fields Virology”, B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). It is one of the most contagious infectious agents known to man and is transmitted from person to person via the respiratory route (Griffin et al., In “Fields Virology”, B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). The measles virus has a complex pathogenesis, involving replication in both the respiratory tract and various systemic sites (Griffin et al., In “Fields Virology”, B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). Measles virus is discussed here as an exemplary pathogen for which a live attenuated vector vaccine is particularly desired. For reasons discussed in further detail herein below, a measles vaccine based on a recombinant HPIV2 vector system would satisfy a long-felt need in the art and fulfill an urgent need for additional effective vector systems to generate vaccines against other pathogens as well.
Although both mucosal IgA and serum IgG measles virus-specific antibodies can participate in the control of measles virus, the absence of measles virus disease in very young infants possessing maternally-acquired measles virus-specific antibodies identifies serum antibodies as a major mediator of resistance to disease (Griffin et al., In “Fields Virology”, B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). The two measles virus glycoproteins, the hemagglutinin (HA) and fusion (F) proteins, are the major neutralization and protective antigens (Griffin et al., In “Fields Virology”, B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996).
The currently available live attenuated measles vaccine is administered by a parenteral route (Griffin et al., In “Fields Virology”, B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). Both the wild type measles virus and the vaccine virus are very readily neutralized by antibodies, and the measles virus vaccine is rendered non-infectious by even very low levels of maternally-acquired measles virus-specific neutralizing antibodies (Halsey et al., N. Engl. J. Med. 313:544-9, 1985; Osterhaus et al., Vaccine 16:1479-81, 1998). Thus, the vaccine virus is not given until the passively-acquired maternal antibodies have decreased to undetectable levels. In the United States, measles virus vaccine is not given until 12 to 15 months of age, a time when almost all children are readily infected with the measles virus vaccine.
Recent developments in the field of negative stranded RNA viral vaccines have involved the use of HPIV3-based vaccine vectors to deliver antigenic determinants of heterologous pathogens, including heterologous HPIVs. In particular, recombinant HPIV3 vaccine candidates have been disclosed that use a HPIV3 “vector” genome or antigenome combined with one or more heterologous genes of a different HPIV, or of a non-PIV pathogen to form a chimeric, bivalent or multivalent, HPIV3-based vaccine candidate (see, e.g., Durbin et al., Virology 235:323-332, 1997; Skiadopoulos et al., J. Virol. 72:1762-1768, 1998; Skiadopoulos et al., J. Virol. 73:1374-1381, 1999; Tao et al., Vaccine 19:3620-3631, 2001; Durbin et al., J. Virol. 74:6821-6831, 2000; U.S. patent application Ser. No. 09/083,793, filed May 22, 1998; U.S. patent application Ser. No. 09/458,813, filed Dec. 10, 1999; U.S. patent application Ser. No. 09/459,062, filed Dec. 10, 1999; U.S. Provisional Application No. 60/047,575, filed May 23, 1997 (corresponding to International Publication No. WO 98/53078), U.S. Provisional Application No. 60/059,385, filed Sep. 19, 1997; U.S. Provisional Application No. 60/170,195 filed Dec. 10, 1999; U.S. patent application Ser. No. 09/733,692, filed Dec. 8, 2000 (corresponding to International Publication No. WO 01/42445A2), each incorporated herein by reference. The recombinant chimeric HPIV3 viruses are engineered to incorporate one or more heterologous donor sequences, typically supernumerary sequences, encoding one or more antigenic determinants of a different PIV or heterologous pathogen to produce an infectious, chimeric, bivalent or multivalent virus or subviral particle. In this manner, candidate HPIV3-based chimeric vaccine viruses can be made to elicit an immune response against one or more PIVs or a polyspecific response against a selected PIV and a non-PIV pathogen in a mammalian host susceptible to infection therefrom. Various modifications to chimeric HPIV3 vaccine candidates were reported to yield desired phenotypic effects, such as attenuation. Comparable disclosure has been provided for recombinant and chimeric recombinant HPIV1 vaccine candidates (see, U.S. Provisional Application No. 60/331,961, filed Nov. 21, 2001, incorporated herein by reference).
Although there have been numerous advances toward development of effective immunogenic compositions against HPIVs and other pathogens, including RSV and measles virus, there remains a clear need in the art for additional tools and methods to engineer safe and effective immunogenic compositions to alleviate the serious health problems attributable to these pathogens, particularly among young infants. Among the remaining challenges in this context is the need for additional tools to generate suitably attenuated, immunogenic and genetically candidates for use in diverse clinical settings against one or more pathogens. Additional challenges arise from the fact that HPIV1, HPIV2, and HPIV3 represent distinct viral serotypes, that do not elicit significant cross-immunity. Accordingly, there is an urgent need in the art for an effective immunogenic compositions to immunize against multiple HPIV serotypes. To facilitate these goals, existing methods for identifying and incorporating attenuating mutations into recombinant strains and for developing vector-based immunogenic compositions and methods must be expanded. In this context, it is particularly desirable to develop a method for recovery and genetic manipulation of HPIV2, to generate immunogenic compositions against this important human PIV, and to provide additional tools to generate novel vectors and immunization methods. Surprisingly, the present invention satisfies these needs and fulfills additional objects and advantages as described herein below.